1. Field of the Invention
The present invention relates to a method of acquiring information of a target object using a time-of-flight secondary ion mass spectrometer and to an imaging detection method by type of a constituent of the target object, in particular, an organic substance such as a protein.
2. Related Background Art
With the developments in recent genomic analyses, there has become important an analysis of proteins which are gene products that exist in a living body, in particular, a protein tip or a technology for visualizing a distributed protein that are present in, e.g., a living tissue.
Conventionally, the importance of analyses of protein expressions and functions has been indicated, and development of the analysis means is proceeding. Basically, the means have been performed by combining:
(1) separation and purification by two-dimensional electrophoresis or high-performance liquid chromatography (HPLC); and
(2) a detection system such as a radiation analysis, optical analysis, or mass spectrometry.
The developments of protein analysis technologies mainly include: database construction by proteome analyses that are bases for the technologies (exhaustive analyses of intracellular proteins); and diagnosis devices or drug discovery (candidate drug screening) devices based on the obtained database. For all application forms, there have been required devices that are different from the conventional devices having problems with analyzing time, throughput, sensitivity, resolution, and flexibility and being suitable for miniaturization, speed enhancement, or automatization. As a means for meeting those requirements, development of a device in which a protein is integrated at high density (so-called protein tip) has attracted attention.
A target molecule captured by a protein tip may be detected by the following various detection means.
In recent years, in the mass spectrometry (MS) method of proteins, time-of-flight secondary ion mass spectrometry (hereinafter abbreviated as TOF-SIMS) has come into use as a sensitive mass analysis means or surface analysis means. The TOF-SIMS is a method of analyzing what atoms or molecules are present on the outermost surface of a solid sample and has the following characteristics. That is, it has an ability to detect ultratrace (109 atoms/cm2) components, can be applied to both organic substances and inorganic substances, enables measurement of all elements or compounds that are present on the surface, and enables imaging of secondary ions from a substance that is present in the surface of a sample.
Hereinafter, the principle of the method will be described briefly.
When high-speed pulsed ion beams (primary ions) are irradiated on the surface of a solid sample in high vacuum, a component of the surface is released into vacuum by a sputtering phenomenon. The generated positively or negatively-charged ions (secondary ions) are focused in one direction by an electrical field, and detection is performed at the position far by a certain distance from there; When pulsed primary ions are irradiated to the solid surface, secondary ions having various masses are generated depending on the composition of the surface of the sample. Among the secondary ions, an ion having a smaller mass flies more speedily, while an ion having a larger mass flies more slowly. Therefore, measurement of a time between generation and detection of the secondary ions (flight time) enables analysis of masses of the generated secondary ions. When primary ions are irradiated, only secondary ions generated at the outermost surface of a solid sample are released into vacuum, so that information about the outermost surface (depth: about a few nm) of the sample can be obtained. In the TOF-SIMS, the amount of irradiated primary ions is significantly small, so that an organic compound is ionized while maintaining its chemical structure, and the structure of the organic compound can be identified from the mass spectra. However, in the case that the TOF-SIMS analysis is performed for an artificial polymer such as polyethylene or polyester, a biological polymer such as a protein, or the like under an usual condition, small degraded fragment ions are generated, so that it is generally difficult to identify the original structure. Meanwhile, in the case that the solid sample is an insulator, the insulator can be analyzed because positive charges accumulated on the solid surface can be neutralized by irradiating pulsed electron beams at the time when pulsed primary ions are not irradiated. In addition, the TOF-SIMS also enables measurement of an ion image (mapping) of the surface of a sample by scanning primary ion beams.
As examples of protein analyses by the TOF-SIMS, the followings are known: detection of a protein parent molecule having a large molecular weight by applying the same pretreatment as the MALDI method, that is, by mixing a protein with a matrix substance (Kuang Jen Wu et al., Anal. Chem., 68, 873 (1996)); imaging detection of a certain protein using secondary ions such as C15N− after labeling a part of the protein of interest with an isotope such as 15N (A. M. Belu et al., Anal. Chem., 73, 143 (2001)); estimation of the kinds of proteins from the kinds of fragment ions (secondary ions) corresponding to amino acid residues or the relative intensities of the fragment ions (D. S. Mantus et al., Anal. Chem., 65, 1431 (1993)); research of the detection limits of the TOF-SIMS for proteins adsorbed on various substrates (M. S. Wagner et al., J. Biomater. Sci. Polymer Edn., 13, 407 (2002)); etc.
Meanwhile, as another mass spectrometry method for proteins, a method utilizing field emission (WO 99/22399) is known. In the method, the above-described proteins are covalently or coordinately bound on a metallic electrode via a cleavable releasing group depending on applied energy and an intense electric field is applied, to thereby lead the above-described proteins to a mass spectrometer.
As described above, for a target object in which plural proteins are dispersedly present, various methods based on the mass spectrometry have been suggested as methods of analyzing the distribution state of the proteins.
However, the conventional mass spectrometry methods are not intended to analyze a target object itself and the resultant information is limited because the methods are directed for an eluted protein or the like. Meanwhile, in the case that the mass spectrometry was performed by the method, it was impossible to directly estimate nonspecific adsorption on the tip surface.
Meanwhile, among ionization methods known today, the MALDI method or the SELDI method that is an improved method thereof is the softest ionization method and has an excellent feature in that it enables ionization of a protein that has a large molecular weight and is easily broken without no additional treatment and enables detection of parent ions or ions based on them. Nowadays, the method is one of standard ionization methods in analyzing the mass of a protein. On the other hand, in the case that those methods are applied to the mass spectrometry with a protein tip, it is difficult to obtain a high spatial resolution two-dimensional distribution image (imaging using mass information) of a protein owing to the existence of a matrix substance. That is, a laser beam itself, which is an excitation source, can be easily condensed to a diameter of about 1 to 2 μm, but the matrix substance that exists around the protein to be analyzed is evaporated and ionized, so that the spatial resolution is generally about 100 μm in the case that the two-dimensional protein distribution image is measured by the above-described method. Meanwhile, to scan the condensed laser, a complex operation for a lens or mirror is required. That is, in the case that a two-dimensional distribution image of a protein is measured by the method, scanning of a laser beam is generally difficult, and there may be employed a system to move a sample stage where a sample to be analyzed is put. In the case of an attempt to obtain a high spatial resolution two-dimensional distribution image of a protein, the system to move the sample stage is generally not preferable.
Moreover, the conventional methods are difficult to provide a two-dimensional distribution image of a target object, and there are limitations in the forms of target samples.
Compared with the above-described methods, the TOF-SIMS method enables easy focusing and scanning because of use of primary ions. Therefore, the method may provide high spatial resolution secondary ion images (two-dimensional distribution images) and also provide a spatial resolution of about 1 μm. However, when the TOF-SIMS measurement is performed for a target object on a substrate under an usual condition, most of the generated secondary ions are small degraded fragment ions, so that it is generally difficult to identify the original structure. Therefore, for a sample such as a protein tip produced by arranging plural proteins on a substrate, any ingenuity is required to obtain high spatial resolution secondary ion images (two-dimensional distribution images) that enable identification of the kind of the proteins of interest. The above-described method by Kuang Jen Wu et al., is a method that enables suppression of degradation of proteins having large molecular weights due to irradiation of primary ions and detection of a parent molecule while maintaining the original mass. However, in the method, a mixture of proteins and a matrix substance is used as a sample to be measured. Therefore, in the case of a sample such as the above-described protein tip, it is impossible to obtain the original two-dimensional distribution information. Meanwhile, the method by A. M. Belu et al., includes labeling a part of a certain protein with an isotope and is a method that enables enough exertion of a high spatial resolution of the TOF-SIMS. However, the labeling of a specific protein with an isotope each time is not general. Meanwhile, in the method shown by D. S. Mantus et al., which is a method of estimating the kinds of proteins from the kinds of fragment ions (secondary ions) corresponding to amino acid residues or relative intensities of the fragment ions, it is difficult to identify the kinds in the case where proteins having similar amino acid compositions exist in a mixture.
Meanwhile, in the case that the TOF-SIMS method is applied to, e.g., a protein molecule in a body tissue, the generation efficiencies of secondary ion species are significantly decreased if maintaining the “holding state” of a peptide chain of the protein molecule. Meanwhile, in measurement using the TOF-SIMS method, a sample to be measured is preliminarily subjected to a drying treatment to perform irradiation of primary ions in high vacuum. In the drying treatment, an interaction occurs between a protein molecule present in a body tissue and another biological substance, and aggregation is caused by intermolecular association, resulting in further lowering of the generation efficiencies of secondary ions.
Therefore, before performing two-dimensional imaging for the abundance distribution of a certain protein molecule in a cutting surface of a body tissue by analyzing the abundance of the certain protein molecule present in the body tissue at high detection sensitivity and high quantification accuracy, it is preferable to preliminarily loosen a peptide chain that constitutes the protein molecule in the “holding state”. Moreover, it is preferable to maintain a state where secondary ion species are generated at high efficiency from an “unholding”peptide chain by suppressing the interaction between a protein molecule and other biological substance. Alternatively, it is preferable to promote or increase generation of secondary ion species from a protein molecule existing in a cutting surface of a body tissue.
Meanwhile, in the TOF-SIMS method, ion sputtering is performed by irradiating primary ions to a molecule to be analyzed, but a difference is caused in the sputtering efficiencies depending on the shape of the surface to be irradiated by the primary ions. As a result, a difference is caused also in the generation efficiencies of secondary ion species derived from the molecule to be analyzed, which may be a trigger to cause a variation in the quantification accuracy. Therefore, it is preferable to suppress the variation also in the generation efficiencies of secondary ion species caused by variation of the shapes of the surfaces to be irradiated by the primary ions. However, conventionally disclosed methods are not necessarily sufficient in those regards.